Project Summary of TR&D 2 ? High-field and fast-MAS SSNMR methods for biomolecular structure and dynamics (PI: Hong) TR&D 2 seeks to develop new solid-state NMR technologies to determine protein structure and dynamics and to produce isotopically labeled cholesterol for membrane protein structural biology. Modern solid-state NMR based structure determination approaches focus on 13C and 15N based resonance assignment and distance measurements. However, the low gyromagnetic ratios of 13C and 15N limit the measurable distances to less than ~8 , which makes it difficult to determine protein three-dimensional folds and quaternary structures. Aim 1 of this TR&D is to overcome this bottleneck in structure determination by developing 19F-based MAS NMR techniques to measure 19F-19F, 19F-13C and 19F-1H distances, potentially to 2 nm. These experiments will be developed for high field and fast MAS conditions to achieve the high-resolution and sensitivity necessary for structure determination. The impact of 19F chemical shift anisotropy on dipolar recoupling will be investigated systematically. Aim 2 of this TR&D is to develop 13C-2H correlation NMR techniques to measure protein dynamics at high magnetic fields with higher angular sensitivity than before. This aim addresses the limitation of 13C-1H and 15N-1H dipolar-coupling based techniques, which are increasingly less sensitive to small- amplitude motions as the magnetic field strength and MAS frequency increase. We will harvest the power of the large 2H quadrupolar coupling for sensing molecular orientations by developing robust polarization transfer techniques between 2H spins and 13C or 15N spins, so that we can measure 13C and 15N resolved 2H quadrupolar spectra. In aim 3 of this TR&D, we will develop biosynthetic methods for high-yield production of 13C and 2H-labeled cholesterol for membrane protein structural biology. By partnering with four driving biomedical projects, we will test and deploy the technologies in this TR&D to address outstanding biological questions in the areas of membrane proteins and cell walls.